1. Field of the Invention
This invention relates to materials for stationary phases in liquid chromatography.
2. Background Information
“High Performance Liquid Chromatography” (HPLC) is a technique first developed in the 1960s (Poole, C.; Poole, S. Chromatography Today, Elsevier, Amsterdam, (1991)) for the separation of a wide variety of analytes that dissolve in solvents. The chromatographic process involves a stationary phase comprising microbeads packed into a separation device, which may include a tube, referred to as a “column”, and a mobile phase that is eluted through the column. An injector introduces a sample carried by a solvent into the column, and a detector senses what is eluted from the column. HPLC stationary phases generally comprise either an inorganic or an organic micro spherical support. In order to increase the surface area per gram and the separation efficiencies, the support is generally formed by an oil/water or a water/oil suspension polymerization of liquid microdroplets of monomers into spherical, porous, solid, micron-sized polymer beads or microbeads. The microbeads are either polymers or copolymers, for example, of styrene and divinylbenzene, and are in the form of a gel. The microbeads may have varying degrees of porosity, and may then be further derivatized to provide a specific surface chemistry. The HPLC gel is packed into steel or heavy-walled plastic columns, allowing the application of high pressures used to force the analyte and a solvent through the column at an increased rate.
A liquid pump, capable of providing uniform flow of from about 0.1 milliliters to about 10 milliliters per minute at pressures of up to about 6,000 pounds per square inch (psi) is often utilized.
Several types of HPLC are used. “Gel permeation chromatography” (GPC), also known as “size exclusion chromatography (SEC)”, or “gel-filtration chromatography” (GFC), is a method of separation in which molecules are separated based on their size, molecular weight, or molecular weight distribution. In SEC, it is intended that the analyte should not have any enthalpic interactions with the stationary phase. The chemical structure of the polymeric supports used to accomplish SEC has historically been dominated by polystyrene divinylbenzene copolymers. Polystyrene divinylbenzene copolymers have also been used as supports in Reverse Phase chromatography.
“Reverse phase” (RP) is a method of separation based on hydrophobic molecular interactions. In RP, separation is accomplished by the differing hydrophobic affinity of the analyte for the column packing material and for the mobile phase or solvent which passes through the column. RP comprises a whole series of related hydrophobic phases including, for example, C18, C8, and C4 bonded to silica, as well as polymeric hydrophobic phases, such as, for example, polydivinylbenzene, polymethylmethacrylate, and polystyrene-polydivinylbenzene copolymers, etc.
“Solid Phase extraction” (SPE) is a method of separation widely used to prepare samples for analysis. Such samples may include, for example, new pharmaceutical compounds, and metabolites in biological fluid matrices such as blood or plasma. Contaminants or other substances such as, for example, proteins in the matrix can interfere with analysis of a compound of interest. In order to determine the concentration of the compound of interest, proteins or other such interfering constituents in the sample can first be removed by using SPE. If the concentration of the compound of interest is high, as the solvent passes through the column, the interfering constituents can be adsorbed by the solid phase while the compound of interest is eluted from the column with the solvent. Alternatively, the compound of interest can be adsorbed by the solid phase while the interfering constituents are washed or eluted through the column. For analysis of complex matrices, both elution strategies can be employed to separate different target analytes.
If the concentration of the compound of interest is too low for detection using any of a variety of analytical detection methods such as mass spectroscopy or ultraviolet detection, with appropriate selection of solvent and solid phase or gel, SPE can also be used to concentrate the compound.
An ongoing problem in the use of SPE is that SPE generally requires a greater difference in selectivity between the compounds to be separated than do other methods of HPLC. In fact, using a currently available solid phase packing material, it has not been possible to prepare some samples for analysis using SPE. This includes compounds which either do not retain on the stationary phase such as some highly water soluble polar drugs or those which cannot be effectively eluted. That is, the currently available solid phase microbeads are inadequate for use in separating the analytes from some samples.
Other common problems in SPE include inadequate resolution of the product from closely related impurities due to incomplete removal of interfering substances from the matrix, and a low rate of recovery of analytes. Further, some samples cannot be prepared for analysis by any currently available HPLC method using available solid phase packing materials. Thus, a need exists for a solid phase polymeric gel that provides a more effective means of separating analytes from a sample.